This invention relates to methods for screening for agents useful for treating hormone disorders, the novel agents identified using such screening methods, and their use to treat hormone disorders.
Millions of people around the world suffer from conditions caused by hormone disorders, including diabetes, pituitary dwarfism and other hypopituitarisms, pituitary gigantism and other hyperpituitarisms, galactorrhea, hypothyroidism (myxedema) and hypothyroidism, a adrenocortical insufficiencies (e.g., Addison""s disease) or hyperfunctions (e.g., Cushing""s syndrome), pheochromocytoma, multiple endocrine neoplasias, polyglandular deficiency syndromes, and disorders of reproductive function.
Diabetes mellitus is a hormone disorder which afflicts millions of people annually. It is a serious and important health problem, involving 2 per cent or more of the US population. It is characterized by an inability to maintain homeostasis of glucose in the bloodstream. Thus the primary symptom of acute diabetes is hyperglycemia. A secondary set of symptoms arises in chronic or long-standing diabetes. These include degeneration of the walls of blood vessels, causing serious vascular complications involving both macro- and microvessels. Many different organs are affected by these complications, and common late clinical manifestations are retinopathy (chronic diabetes is a leading cause of blindness), nephropathy, neuropathy and foot ulcers.
In normal individuals, an increase in glucose concentration in the blood (xe2x80x9cblood glucosexe2x80x9d) triggers the release of insulin from the pancreas into the bloodstream. This in turn leads to uptake of glucose into tissues and its conversion into glycogen and fatty acids. In most diabetic individuals there is a deficiency in the production, release, stability or uptake of insulin. This results in an inability to remove glucose from the bloodstream and to store its fuel content by metabolizing it into glycogen and fatty acids. Even in the presence of elevated blood glucose, the metabolism of a diabetic is geared towards the synthesis of glucose via gluconeogenesis and the oxidative breakdown of fatty acids.
Many other hormone disorders are characterized by an under- or over-abundance of the hormone in question, due to abnormalities in its synthesis, release or rate of elimination from the bloodstream, and/or by the inability of target cells to respond normally to the hormone, due to abnormalities in the number or function of receptors for the hormone or in the signal transduction pathways which mediate cells"" responses to the hormone.
Applicant has determined that certain cellular components interact with translation factors in the cell and modulate cellular translation in response to a hormone. To this end, Applicant believes that agents which mimic this activity will be useful for treatment of hormone deficiencies. Applicant now provides a means to identify such useful agents. Thus, the present invention relates to methods for screening for agents useful to treat hormone disorders. The screening methods utilize a protocol in which potentially useful non-hormone agents are brought into contact with a system containing a cellular component and a translation factor which interact with one another in intact cells in a manner that is normally responsive to the hormone in question and results in a modulation of translation in the cell. The impact of the test agents on the interaction between the cellular component and the translation factor is then determined. Those agents which modify this interaction may be useful for the treatment of hormone disorders. Once identified, such agents can be formulated in therapeutic products (or even prophylactic products) in pharmaceutically acceptable formulations, and used for specific treatment of hormone disorders with few side effects. While such agents may be useful as hormone substitutes, they are also useful in test systems to allow an understanding of the action of the hormone.
Thus, in a first aspect, the invention features a method for screening for an agent useful to treat a hormone disorder by contacting a potential agent with a system containing a cellular component and a translation factor. The component and the factor have the property that they interact with one another in intact cells in a manner that is normally responsive to the hormone and results in a modulation of translation. The method also includes determining whether the agent causes a modification of any interaction between the component and the factor similar to any modification that would normally occur in intact cells in response to the hormone.
By xe2x80x9cscreeningxe2x80x9d is meant a process in which a large number of potentially useful agents are processed in the method of this invention. It is a process distinct from a single experiment in which a single agent is studied in detail to determine its method or mode of action.
By xe2x80x9chigh-throughput screeningxe2x80x9d is meant screening in which many potentially useful agents can be processed in a short period of time. By way of example, hundreds of the agents might be processed in a single day, or thousands in a single week.
By xe2x80x9chormone disorderxe2x80x9d is meant any human or animal disease or condition caused by or characterized by abnormally high or low concentrations or availability of a hormone.
By xe2x80x9cabnormally highxe2x80x9d is meant any difference above normal sufficient to be manifested by physiological, biochemical, physical, mental, or psychological effects.
By xe2x80x9cabnormally lowxe2x80x9d is meant any difference below normal sufficient to be manifested by physiological, biochemical, physical, mental, or psychological effects.
By xe2x80x9ccellular componentxe2x80x9d is meant any component found within a cell. Such components include, but are not limited to, proteins, lipoproteins, glycoproteins, lipids, carbohydrates, nucleic acids, steroids, prostaglandins, and combinations and complexes thereof.
By xe2x80x9ctranslation factorxe2x80x9d is meant a molecule or group of molecules which participates directly at some stage in the process of translation but which is not permanently attached to or associated with the ribosome. Such factors include, but are not limited to, eIF-1, eIF-2 and its three known constituent polypeptides, eIF-2B (also known as GEF) and its five known constituent polypeptides, eIF-3 and its eight known constituent polypeptides, eIF-4A, eIF-4B, eIF-4C (also known as eIF-1A), eIF-4D (also known as eIF-5A), eIF-4E, eIF-4F and its three known constituent polypeptides, eIF-5, eIF-6 (also known as eIF-3A), eEF-1xcex1, eEF-1xcex2xcex3 and its two known constituent polypeptides, eEF-2, and eRF.
By xe2x80x9cinteract with one anotherxe2x80x9d is meant that the entities in question become physically associated with one another, transiently or for longer periods, or that one causes a biochemical or conformational change in the other.
By xe2x80x9cnormally responsive to the hormonexe2x80x9d is meant that the molecule, molecules, complex or process in question undergoes some physical or biochemical alteration as a result of the presence, the absence, or a change in concentration of the hormone in question.
By xe2x80x9chormonexe2x80x9d is meant any molecule or group of molecules released by a cell or cell type which has a physical or biochemical effect on the same or another cell or cell type. Such molecules include, but are not limited to, peptides and protein hormones such as insulin, glucagon, vasopressin, calcitonin, ACTH, growth hormone and the like, steroid hormones such as estrogen, testosterone and the like, and growth factors such as platelet-derived growth factor, epidermal growth factor, nerve growth factor, interleukins, other cytokines, and the like. Non-hormones are other components or other chemicals or biochemicals, as described below in xe2x80x9cLibraries.xe2x80x9d
By xe2x80x9cmodulation of translationxe2x80x9d is meant a control, or change of control, of the efficiency or rate of translation of mRNAs which results in a change in the overall rate of protein synthesis, the relative quantities of different proteins synthesized, and/or the quantities of individual proteins synthesized.
By xe2x80x9cmodification of any interaction between the component and the factorxe2x80x9d is meant a change in the quantity of either participant involved in the interaction, in the relative quantities of the participants, in the affinity, stability or strength of their interaction, or in the rate constants for their association, dissociation, or alteration of one another.
A modification xe2x80x9csimilar to any modification that would normally occur in intact cells in response to the hormonexe2x80x9d is a modification in which the change observed (as defined in the previous paragraph) involves the same participant or participants, and is of the same type and order of magnitude as the corresponding that normally occurs in intact cells in response to the hormone. Thus a parameter which normally increases in intact cells should increase in the system, and a parameter that normally decreases in intact cells should decrease in the system. Insofar as such a comparison can be performed, the extent of the increase or decrease in the system should preferably be at least 10%, preferably at least 25%, and most preferably at least 50% of the corresponding increase or decrease that normally occurs in intact cells in response to the hormone.
In preferred embodiments, the hormone disorder is diabetes mellitus and the hormone is insulin.
In further preferred embodiments, the cellular component which interacts with the translation factor is a protein which becomes phosphorylated in intact cells in response to the hormone.
By xe2x80x9cphosphorylatedxe2x80x9d is meant that one or more phosphate groups is added to the protein, which may or may not already carry one or more phosphate moieties prior to the addition of the phosphate groups.
In further preferred embodiments, the modification in the interaction between the component and the factor is a reduction of the interaction. In more preferred embodiments, the reduction results in a stimulation of translation.
By xe2x80x9creduction of the interactionxe2x80x9d is meant any reduction which can be detected in the absolute or relative quantity of either participant involved in the interaction, or in the affinity, stability or strength of their interaction, or in the rate constants for their association or their alteration of one another. Such a reduction is preferably at least two-fold, more preferably at least three-fold, and most preferably at least five-fold.
By xe2x80x9cstimulation of translationxe2x80x9d is meant any increase in the efficiency or rate of translation of mRNA which results in an increase in the overall rate of protein synthesis, in the relative quantities of different proteins synthesized, and/or in the quantities of an individual protein or proteins synthesized. Such an increase is preferably at least two-fold, more preferably at least three-fold, and most preferably at least five-fold.
In more preferred embodiments, the component is 4E-BP1, the factor is eIF-4E, and the system is an in vitro translation system in which the interaction between the 4E-BP1 and the eIF-4E is detected by translation of RNA sequences encoding one or more reporter polypeptides. More preferably, the RNA is capped and/or contains a 5xe2x80x2-untranslated region (5xe2x80x2-UTR) with significant secondary structure. More preferably, one of the reporter polypeptides in the enzyme luciferase.
By xe2x80x9c4E-BP1xe2x80x9d is meant the protein of 118 amino acids whose amino acid sequence is provided in FIG. 1, and which is encoded by the nucleic acid sequence therein. This protein has a molecular weight of about 12 kilodaltons (kD) but migrates anomalously during SDS/polyacrylamide gel electrophoresis, with an apparent molecular weight of approximately 22 kD. The protein binds to eIF-4E and causes inhibition of translation. Phosphorylation of this protein, for example in response to insulin, causes its release from eIF-4E and a consequent stimulation of translation.
By xe2x80x9ceIF-4E xe2x80x9d is meant the 25 kD protein also known as cap-binding protein which is a subunit of eIF-4F (also known as eIF-4), and whose function is to bind the cap at the 5xe2x80x2-end of mRNA molecules and assist in the association of these molecules with ribosomes at the initiation stage of translation.
By xe2x80x9cin vitro translation systemxe2x80x9d is meant a cell-free extract capable of translating a protein or proteins from an RNA or RNAs encoding such proteins. Such a mixture typically contains ribosomes, tRNAs, amino acids, salts, and various other factors required to sustain protein synthesis, in addition to the RNA(s) that direct protein synthesis. Such mixtures are typically prepared from sources such as, but not limited to, rabbit reticulocytes, HeLa cells, wheat germ, and E coli cells; extracts prepared from such sources may be supplemented by the addition of tRNAs, amino acids, and so on, as necessary.
By xe2x80x9creporter polypeptidexe2x80x9d is meant any protein which can be detected and measured by a physical, biochemical, enzymatic, immunological or other procedure.
By xe2x80x9c5xe2x80x2-untranslated region with significant secondary structurexe2x80x9d is meant a 5xe2x80x2-UTR which contains at least one sequence at least 30 nucleotides in length capable of forming by intrastrand base-pairing a second structure which impedes movement of a ribosome or ribosomal subunit along the RNA containing the 5xe2x80x2-UTR. The sequence should preferably begin within 50 nucleotides of the 5xe2x80x2-end of the RNA, and form a secondary structure with a free energy of at least xe2x88x921 kcal/mol, preferably at least xe2x88x922 kcal/mol, most preferably at least xe2x88x925 kcal/mol for every ten nucleotides involved in the secondary structure.
By xe2x80x9cluciferasexe2x80x9d is meant any polypeptide, protein or part of a protein capable of catalyzing the conversion of luciferin to oxyluciferin with concomitant release of light. Examples of luciferases include, but are not limited to, the luciferase enzyme of the firefly Photinus pyralis, which has a molecular weight of approximately 65 kilodaltons.
In highly preferred embodiments, a modification to the interaction between the eIF-4E and the 4E-BP1 is detected by comparing translation of a first reporter polypeptide whose translation is dependent on eIF-4E with translation of a second reporter polypeptide whose translation is not dependent on eIF-4E. Preferably, the coding sequence for the second reporter polypeptide is translationally linked to an IRES. The coding sequences for the first and the second reporter polypeptides may be contained in separate RNAs, or in a bicistronic RNA, that is, an RNA containing coding sequences for two polypeptides. Said bicistronic RNA preferably contains an IRES between the coding sequences.
By xe2x80x9ctranslation dependent on eIF-4Exe2x80x9d is meant translation which requires eIF-4E for its initiation. Since a key role of eIF-4E is to bind to the cap structure found at the 5xe2x80x2-ends of eucaryotic mRNAs, translation of capped RNAs is dependent on eIF-4E. In in vitro translation systems mRNAs can usually be translated even if they are not capped. Such translation of uncapped mRNAs may nevertheless be dependent on eIF-4E, whose interaction with the other subunits of eIF-4 may be essential for the helicase activity of the eIF-4 which xe2x80x9cmelts secondary structure at and near the 5xe2x80x2-ends of mRNAs. In any specific case, it can be determined whether the translation of a particular mRNA is dependent on eIF-4E by determining whether this translation is reduced in the presence of additional 4E-BP1 or 4E-BP2.
By xe2x80x9c4E-BP2xe2x80x9d is meant the protein of 120 amino acids whose amino acid sequence is provided in FIG. 2, which is encoded by the nucleic acid sequence shown therein. This protein has a molecular weight of about 12 kilodaltons""s (kD) but migrates anomalously during SDS/polyacrylamide gel electrophoresis, with an apparent molecular weight of approximately 20 kD. The protein binds to eIF-4E and causes inhibition of translation.
By xe2x80x9ctranslation not dependent on eIF-4Exe2x80x9d is meant translation which does not require eIF-4E for its initiation. A good example is translation which is initiated at an IRES. In any specific case, it can be determined whether translation of a particular mRNA is not dependent on eIF-4E by determining that this translation is not significantly reduced in the presence of additional 4E-BP1 or 4E-BP2.
A coding sequence which is xe2x80x9ctranslationally linkedxe2x80x9d to an IRES is one which depends on the IRES for its translation. Typically, this means that the coding sequence is contained on same RNA molecule as the IRES and that the IRES is positioned adjacent to, and closer to the 5xe2x80x2-end than, the coding sequence.
By xe2x80x9cIRESxe2x80x9d is meant internal ribosome entry site, that is, a site within a RNA molecule which allows translation of that RNA to be initiated by the binding of a ribosome or subunit internally rather than at the cap structure the 5xe2x80x2-end. Such initiation can therefore take place independently of eIF-4E, and the activity of eIF-4E can be assessed by comparing translation of a protein which depends on cap-dependent initiation with translation of a protein which depends on IRES-mediated initiation. Preferably, the IRES is derived from the IRES region in the 5xe2x80x2-untranslated region (UTR) of a picornvirus RNA; the IRES is selected from the group consisting of enterovirus, rhinovirus, cardiovirus, aphthovirus, hepatitis A virus, hepatitis B virus and hepatitis C virus IRES sequences.
By xe2x80x9cbicistronic RNAxe2x80x9d is meant a RNA containing the coding sequences for two polypeptides. Preferably, translation of the first coding sequence, that is, the coding sequence closer to the 5xe2x80x2-end, is dependent on normal cellular initiation processes requiring eIF-4E, and translation of the other coding sequence is dependent on IRES-mediated initiation processes. Said bicistronic RNA may not need to be capped in order that the translation of the first coding sequence be eIF-4E-dependent. In the case of an uncapped bicistronic RNA, it can be determined whether the translation of the first coding sequence is dependent on eIF-4E by determining whether this translation is reduced in the presence of additional 4E-BP1 or 4E-BP2.
In further preferred embodiments, the determination of whether the agent causes a modification of an interaction between the component and the factor comprises measurement of binding of the component to the factor. In a more preferred embodiment, one of the component and the factor is attached to a solid phase and the other of the component and the factor is detected using an easily detectable reporter. Preferably, the component is 4E-BP1 and the factor is eIF-4E. Most preferably, the binding of the component to the factor is measured by measuring a protein translated in a cell from a RNA whose transcription depends on the binding.
By xe2x80x9cbindingxe2x80x9d is meant a physical association between two or more molecules which is more prolonged and/or of greater strength or affinity than would be observed following random collisions of molecules that do not bind to one another. Such binding may be transient or for a longer period.
By easily detectable reporter is meant any agent or substance which can be readily detected by physical, chemical, biochemical, enzymatic or other means. Such reporters include, but are not limited to, enzymes, fluorescent, luminescent, or chromophoric molecules, antibodies labeled with any of the foregoing, and haptens and antigens that can be detected using such antibodies.
In a second aspect the invention provides a method for screening for an agent useful to treat a hormone disorder, comprising the steps of contacting a potential the agent with a system containing an enzyme which modifies a cellular component in a manner that is normally responsive to the hormone and results in a modulation of translation, and determining whether the agent causes an increase or decrease in the activity of the enzyme.
By xe2x80x9can enzyme which modifies a cellular componentxe2x80x9d is meant an enzyme which catalyzes a chemical change of that component. Such chemical changes may include, but are not limited to, the addition or removal of phosphate groups, carbohydrate moieties, lipid moieties, nucleotidyl moieties, and the like; and oxidations, reductions, dehydrogenation, hydrogenations, and the like.
In preferred embodiments, the enzyme which modifies the cellular component is a kinase.
By xe2x80x9ckinasexe2x80x9d is meant an enzyme which adds a phosphate group or phosphate groups to the component, which may or may not already contain phosphate moieties prior to the addition of the phosphate group or groups.
In preferred embodiments, the enzyme which modifies the cellular component is a phosphatase.
By xe2x80x9cphosphatasexe2x80x9d is meant an enzyme which removes a phosphate group or phosphate groups from the component, which may or may not retain other phosphate moieties following the removal of the phosphate group or groups.
In additional preferred embodiments, the hormone is insulin and the component is 4E-BP1.
In a third aspect, the invention features a method of treating a patient with a hormone disorder by administering, in a therapeutically effective dose, a pharmaceutically acceptable formulation of an agent which causes a modification of an interaction between a cellular component and a translation factor, where the component and the factor have the property that they interact with one another in intact cells in a manner that is normally responsive to the hormone and results in a modulation of translation.
By xe2x80x9ctherapeutically effective dosexe2x80x9d is meant an amount that relieves (to some extent) one or more symptoms of the disease or condition in the patient. Additionally, by xe2x80x9ctherapeutically effective dosexe2x80x9d is meant an amount that returns to normal, either partially or completely, physiological or biochemical parameters associated with or causative of a hormone disorder. Generally, it is an amount between about 1 nmole and 1 xcexcmole of the molecule, dependent on its EC50 and on the age, size, and disease associated with the patient.
By xe2x80x9cpharmaceutically acceptable formulationxe2x80x9d is meant a pharmaceutical composition prepared for storage and subsequent administration, which comprise a pharmaceutically effective amount of an agent as described herein in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington""s Pharmaceutical Sciences, Mack Publishing Go. (A. R. Gennaro edit. 1985). Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives. Id. at 1449. In addition, antioxidants and suspending agents may be used. Id The invention features agents formulated for use in treatment of hormone disorders, and any such unique (i.e., previously unknown) formulations are within this invention.
In preferred embodiments, the invention features methods of treating a patient with diabetes by administering an agent effective to reduce the interaction between the component and the factor. In more preferred embodiments, the component is 4E-BP1 and the factor is eIF-4E.
In a seventh aspect, the invention features a purified cellular component that binds to eIF-4E and causes a modulation of translation in a cell in response to a hormone.
In preferred embodiments, the component comprises the protein 4E-BP1 or the protein 4E-BP2.
In an eighth aspect, the invention provides a purified nucleic acid sequence encoding a cellular component that binds to eIF-4E and causes a modulation of translation.
By purified is meant that the protein or nucleic acid is separated from the environment in which it naturally occurs, and is preferably in a homogeneous form suitable for use in test assays as described herein. Such components are useful in the assays described below.
In preferred embodiments, the sequence codes for the protein 4E-BP1 or for the protein 4E-BP2.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.